Amiloride-sensifive sodium transport is a hallmark of many sodium-transporting epithelial tissues where it plays a key role in regulation of total body salt and fluid balance. Transport is achieved by a two step process involving movement of sodium from the lumen into the epithelial cell interior through amiloride-inhibitable sodium channels and subsequent active extrusion of sodium into the serosal space by the basolateral sodium pump. Under most circumstances, the overall transport rate is determined by the activity of the apical channels so their regulation is important for determining total body sodium balance. Since in all sodium-transporting tissues examined to date, molecular biological studies have confirmed the presence of amiloride-sensitive epithelial Na+ channels (ENaC) subunits, a, [3,and y, it has generally been assumed that apical Na +transport is mediated by some form of ENaC. Functional epithelial sodium channels (ENaC) are formed by the assembly of some combination of the three subunits into a tetrameric structure. This process presumably occurs within the endoplasmic reticulum (ER) and is inefficient, as only a fraction of newly synthesized ENaC subunits assemble into channels that exit the ER and reach the plasma membrane. Our preliminary experiments show that both cells transfected with ENaC subunits and native epithelial cells can express three distinct types of cation channels that are capable of transporting Na +. These highly selective, medium-selective, and non-selective cation channels appear to be composed of different combinations of ENaC subunits. Thus, unlike multimeric cation channels in excitable tissues which are eotranslationally assembled, amiloride-sensitive cation channels appear to be post-translationally assembled and under the permissive conditions some, but not all subunits can traffic to the plasma membrane. This raises the natural set of questions of how individual ENaC subunits are trafficked, where the subunits are assembled into functional ion channels, how they are inserted into the surface membrane, and how they are retrieved and degraded or recycled. In addition, a second set of questions is how these processes are regulated to produce the hormone-induced changes in the number and type of functional channels in the apical membrane. These questions form the basis for the hypotheses and specific aims of this project.